Docstoc

BIOLOGICAL MEMBRANES - Department of Biochemistry _ Molecular

Document Sample
BIOLOGICAL MEMBRANES - Department of Biochemistry _ Molecular Powered By Docstoc
					            BIOLOGICAL MEMBRANES
!   Overview
     biological roles
     structural features

!   Membrane lipids
       general structures
       aggregation states
       polymorphism
       thermal transitions
       electrical conductivity
       electrostatic effects
       molecular dynamics (translational and rotational diffusion,
             flip-flop)

!   Membrane proteins
     crystallization
     overview of structural features
     structure/function relations:
           bacterial photosynthetic reaction center
           bacteriorhodopsin
             Biochemistry 585; Membrane Proteins
                         Reading List


                       CRYSTALLIZATION

*C.Ostermeier & H. Michel, ACrystallization of membrane
proteins@, Curr. Opinion Struct. Biol. 7, 697-701 (1997).

#C.Ostermeier, S. Iwata, B. Ludwig & H. Michel, AFv fragment-
mediated crystallization of the membrane protein bacterial
cytochrome c oxidase@, Nature Structural Biology, 2, 842-846
(1995).

Optional: *E.M. Landau & J.P. Rosenbusch, ALipidic cubic
phases: A novel concept for the crystallization of membrane
proteins@, Proc. Natl. Acad. Sci. USA 93, 14532-14535 (1996).

[ *pdf files on website; #reprint will be provided ]
              STRUCTURES AND FUNCTIONS

        Overview:

*S.
  Scarlata, "Membrane Protein Structure", Chap. 1,
Section 2, Biophysical Society on-line textbook.

*J.U.Bowie, "Membrane proteins: are we destined to
repeat history", Curr. Opinion Struct. Biol. 10, 435-437
(2000).

*G.G. Shipley, "Lipids; Bilayers and non-bilayers:
structures, forces and protein crystallization", Curr.
Opinion Struct. Biol. 10, 471-473 (2000).
      Electron Transfer Mechanisms:

Optional:
*J.R. Winkler, "Electron tunneling pathways in proteins",

Curr. Opinion in Chem. Biol. 4, 192-198 (2000).

#C.C. Page, C.C. Moser X. Chen & P.L. Dutton, "Natural
engineering principles of electron tunneling in biological
oxidation-reduction", Nature 402, 47-52 (1999).
        Bacterial Photosynthetic Reaction Center:

#U. Ermler, H. Michel & M. Schiffer, "Structure and function
of the photosynthetic reaction center from Rhodobacter
sphaeroides", J. Bioenerg. Biomembr. 26, 5-15 (1994).
*J.P.
    Allen & J.C. Williams, "Photosynthetic reaction
centers", Minireview, FEBS Lett. 438, 5-9 (1998).

#N.W.  Woodbury & J.P. Allen, APathway, kinetics and
thermodynamics of electron transfer in wild type and
mutant reaction centers of purple nonsulfur bacteria@, in
Anoxygenic Photosynthetic Bacteria, R.E. Blankenship et
al., eds, Chap. 24, pp. 527-557, Kluwer Acad. Publ., 1995.
Optional:
*J. Deisenhofer et al., ACrystallographic refinement at 2.3 D

resolution and refined model of the photosynthetic
reaction centre from Rhodopseudomonas viridis@, J. Mol.
Biol. 246, 429-457 (1995)].
*P.K.Fyfe and M.R. Jones, "Re-emerging structures:
continuing crystallography of the bacterial reaction
centre", Biochim. Biophys. Acta 1459, 413-421 (2000).

*M.Y.Okamura et al., "Proton and electron transfer in
bacterial reaction centers", Biochim. Biophys. Acta 1458,
148-163 (2000).]
        Bacteriorhodopsin:

    Lanyi and H. Luecke “Bacteriorhodopsin”, Curr.
*J.K.

Opinion Struct. Biol., 11, 415-419 (2001).

#W. Khlbrandt "Bacteriorhodopsin- the movie", Nature
406, 569-570 (2000).

Optional:
*J.K. Lanyi “Bacteriorhodopsin”, Bioenergetics, Chap. 3,

Biophysical Society on-line textbook.
       BIOLOGICAL ROLES OF MEMBRANES

 SELECTIVE PERMEABILITY BARRIERS (CELL
COMPARTMENTALIZATION): PUMPS, GATES SIEVES

 STRUCTURAL ORGANIZATION OF CELLULAR
PROCESSES (ENERGY TRANSDUCTION): RESPIRATION,
PHOTOSYNTHESIS, VISION
 RECEPTORS FOR EXTERNAL STIMULI: HORMONES,
NEUROTRANSMITTERS
 CELL RECOGNITION: IMMUNE RESPONSE, TISSUE
FORMATION
 INTERCELLULAR COMMUNICATION: NERVE IMPULSE
TRANSMISSION
 MOST MEMBRANES ARE MULTI-FUNCTIONAL
             STRUCTURAL FEATURES OF MEMBRANES
 MULTIPLE COMPONENTS
      LIPIDS (PHOSPHOLIPIDS, GLYCOLIPIDS, CHOLESTEROL):
             BILAYER STRUCTURE FORMS MAIN PERMEABILITY
                   BARRIER.
      PROTEINS (PERIPHERAL, INTEGRAL): PROVIDE BOTH
             STRUCTURAL AND FUNCTIONAL CHARACTERISTICS.
      CARBOHYDRATE (COVALENTLY BOUND TO LIPID AND
             PROTEIN): SURFACE RECOGNITION.
 BROAD COMPOSITIONAL VARIABILITY
      CORRELATED WITH FUNCTION
 MOSTLY SELF ASSEMBLING
      HYDROPHOBIC AND ELECTROSTATIC FORCES LEAD TO
      BILAYER FORMATION AND PROTEIN INCORPORATION
      (CARBOHYDRATE ADDED ENZYMATICALLY AFTER ASSEMBLY)
 ASYMMETRIC
     INSIDE DIFFERENT FROM OUTSIDE WITH RESPECT TO LIPID AND
     PROTEIN (CARBOHYDRATE ONLY FOUND ON OUTER SURFACE)
 DYNAMIC STRUCTURE
      FLUIDITY, FLEXIBILITY, TWO-DIMENSIONAL DIFFUSION
BIOLOGICAL SIGNIFICANCE OF LIPID POLYMORPHISM

POTENTIAL TO FORM NONBILAYER STRUCTURES MAY
ALLOW DISCONTINUITIES IN BILAYER AND THEREBY
PROMOTE:

  MEMBRANE FUSION AND VESICLE FORMATION
     DURING CELL DIVISION.
  VESICLE-MEDIATED PROTEIN TRAFFICKING.
  INTEGRATION OF NON-LIPID COMPONENTS INTO
     MEMBRANE.
  MOVEMENT OF MACROMOLECULES THROUGH
     MEMBRANE.
  LATERAL MOVEMENT OF MACROMOLECULES.
  STABILIZATION OF MEMBRANE PROTEIN
     COMPLEXES.
  CONFORMATIONAL INTERCONVERSIONS
     ASSOCIATED WITH PROTEIN FUNCTION.
      TRANSLATIONAL DIFFUSION IN MEMBRANES
 USUALLY MEASURED BY FRAP (FLUORESCENCE RECOVERY
     AFTER PHOTOBLEACHING) USING FLUOROPHORE-
     LABELLED LIPIDS.
 INVOLVES PHOTOBLEACHING A SMALL REGION OF
       MEMBRANE SURFACE WITH LASER AND MEASURING
       TIME DEPENDENCE OF MOLECULAR DIFFUSION INTO
       BLEACHED AREA.
 Dtrans (translational diffusion coefficient) RELATED TO
       MEAN SQUARE DISPLACEMENT:
                   _
                   r2  4 Dtrans t
 FOR BOTH LIPIDS AND PROTEINS, Dtrans  10-8 cm2s-1 at 25 °C.
      THUS, IN 1 SECOND:
       _
       r2 = 4 x 10-8 cm2
      _
      (r2)1/2 (MEAN DISPLACEMENT) = 2 x 10-4 cm = 2 microns
              (i.e. MOVEMENT IS RAPID).
 MEASUREMENT OF MEMBRANE FLUIDITY AND MOLECULAR
      ROTATION BY FLUORESCENCE DEPOLARIZATION
USE A COVALENTLY ATTACHED FLUOROPHORE, OR A
      FLUORESCENT PROBE WHICH PARTITIONS INTO THE
      BILAYER (e.g. DPH; DIPHENYLHEXATRIENE). EXCITE WITH
      POLARIZED LIGHT AND MEASURE POLARIZATION OF
      FLUORESCENCE. IF FLUOROPHORE ROTATES DURING
      EXCITED STATE LIFETIME, FLUORESCENCE WILL BECOME
      DEPOLARIZED.
DEFINITIONS:
            P = POLARIZATION = (I - I) / (I + I)

           r = ANISOTROPY = (I - I) / (I + 2I)
PERRIN EQUATION:
            r0 / r = DEGREE OF DEPOLARIZATION = 1 + (F / C)

WHERE:
              r0 = ANISOTROPY IN RIGID MATRIX (I.E. NO ROTATION)
              r = ANISOTROPY IN MEMBRANE
                F = FLUORESCENCE LIFETIME
                C = ROTATIONAL CORRELATION TIME = 1 / DROT
                       DROT = ROTATIONAL DIFFUSION COEFFICIENT
  PERRIN EQUATION ALLOWS ROTATIONAL CORRELATION TIME TO
  BE DETERMINED. THIS CAN BE RELATED TO SOLVENT VISCOSITY
  (FOR A SPHERICAL MOLECULE) BY:
              c = V / k T

  where:
                = VISCOSITY
               V = VOLUME OF FLUOROPHORE

  USUALLY USE A CALIBRATION CURVE TO CALCULATE
  MICROVISCOSITY OF MEDIUM. IN GENERAL:
               lipid bilayer  100  water

CAN ALSO BE APPLIED TO PROTEINS IN A MEMBRANE TO OBTAIN
       DROT. FOR TWO-DIMENSIONAL ROTATIONAL MOTION:
                   Drot = k T / 4  a2 h 
FOR A "TYPICAL" MEMBRANE PROTEIN: Drot = 105 s-1; c = 2 s

                                 h
                             a
       ELECTROSTATIC EFFECTS AT MEMBRANE SURFACES
 MEMBRANE SURFACE CHARGE WILL INFLUENCE LOCAL
CONCENTRATIONS OF CHARGED SPECIES, INCLUDING HYDROGEN
IONS, SALT IONS AND PROTEINS.
 THE SURFACE POTENTIAL OF A MEMBRANE CAN BE
CALCULATED FROM ELECTROSTATIC DOUBLE LAYER THEORY
(GUOY-CHAPMAN THEORY; cf. CEVC & MARSH, “PHOSPHOLIPID
BILAYERS”, WILEY-INTERSCIENCE, 1987).
      (in mV) = (2kT/Ze) ln (0.36 Ac C1/2)
      Z = charge valency of counterions
      Ac = area per charge at membrane surface (in nm2)
      C = molar concentration of salt ions
 FROM THIS POTENTIAL, ONE CAN CALCULATE THE LOCAL
CONCENTRATION OF A CHARGED PROTEIN, AND THE LOCAL pH:
          [P]surface = [P]bulk exp(-Z  / kT)
           where Z is the net protein charge.
          pHsurface = pHbulk + e  / 2.3 kT
NOTE THAT  IS ALWAYS NEGATIVE FOR BIOMEMBRANES. ALSO,
BOTH OF THESE QUANTITIES WILL BE STRONGLY AFFECTED BY SALT
CONCENTRATION.
HIGH RESOLUTION MEMBRANE PROTEIN CRYSTAL STRUCTURES (as
of March, 2002) [~17,000 soluble protein structures listed]
1- Porin: M.S. Weiss & G.E. Schulz, J. Mol. Biol. 227, 493-509 (1992).

2- Bacterial photosynthetic reaction center: J. Deisenhofer et al., J. Mol.
Biol. 246, 429-457 (1995).

3- Prostaglandin synthase: D. Picot et al., Nature 367, 243-249 (1994).

4- Cytochrome c oxidases: S. Iwata et al., Nature 376, 660-669 (1995); T.
Tsukihara et al., Science 272, 1136-1144 (1996); Soulimane et al., EMBO
J. 19, 1766-76 (2000).

5- Bacterial light-harvesting complex: G. McDermott et al., Nature 374,
517-521 (1995); J. Koepke et al., Structure 4, 581-597 (1996).

6- -Hemolysin: L. Song et al., Science 274, 1859-1866 (1996).

7- Cytochrome bc1: D. Xia et al., Science 277, 60-66 (1997); Z. Zhang et
al., Nature 392, 677-684 (1998); S. Iwata et al., Science 281, 64-71 (1998);
C. Hunte et al., Structure 8, 669-684 (2000).
8- Bacteriorhodopsin: H. Luecke et al., J. Mol. Biol. 291, 899-911 (1999)

9- Potasssium ion channel: D.A. Doyle et al., Science 280, 69-77 (1998).

10- Iron transport protein (FhuA): A.D. Ferguson et al., Science 282,
2215-2220 (1998).

11- Mechanosensitive ion channel (MscL): G. Chang et al., Science 282,
2220-2226 (1999).

12- Fumarate reductase: T.M. Iverson et al., Science 284, 1961-1966
(1999); C.R.D. Lancaster et al., Nature 402, 377-385 (1999).

13- Outer membrane active transporter (FepA): S.K. Buchanan et al.,
Nature Struct. Biol. 6, 56-63 (1999).

14- Squalene-hopene cyclase: K.U. Wendt et al., J. Mol. Biol. 286, 175-
187 (1999).

15- Outer membrane phospholipase A: H.J. Snijder et al., Nature 401,
717-721 (1999).
16- Sarcoplasmic reticulum calcium pump: Toyoshima et al., Nature
405, 647-655 (2000).

17- E. coli glycerol channel: Fu et al. Science 290, 481-486 (2000).

18- Rhodopsin: Palczewski et al., Science 289, 739-745 (2000).

19- Halorhodopsin: Kolbe et al., Science 288, 1390-1396 (2000).

20- TolC outer membrane pore: Koronakis et al., Nature 405, 914-919
(2000).

21- Sensory Rhodopsin: Leucke et al., Science 293, 1499-1503 (2001);
Royant et al., PNAS 98, 10131-10136 (2001).

22- Photosystem I: Jordan et al., Nature 411, 909-917 (2001); Barber,
Nature Struct. Biol. 8, 577-579 (2001).

23- Photosystem II: Zouni et al., Nature 409, 739-743 (2001).

24- C1C chloride channel: Dutzler et al., Nature 415, 287-294 (2002)
23- Formate dehydrogenase: Jormakka et al., Science 295, 1863-1869
(2002).

Web site:
       http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
CURRENT OPINION IN STRUCTURAL BIOLOGY, 7, 697-701 (1997).
Crystallization of membrane proteins
Christian Ostermeier* and Hartmut Michel†
         Five new membrane protein structures have been
determined since 1995 using X-ray crystallography: bacterial
light-harvesting complex; bacterial and mitochondrial
cytochrome c oxidases; mitochondrial bc 1 complex; and
a-hemolysin. These successes are partly based on advances
in the crystallization procedures for integral membrane
proteins. Variation of the size of the detergent micelle and/or
increasing the size of the polar surface of the membrane
protein is the most important route to well-ordered membrane
protein crystals. The use of bicontinuous lipidic cubic phases
also appears to be promising.
Addresses
* Department of Molecular Biophysics and Biochemistry, Yale
University, Bass Center 433, Whitney Avenue, New Haven,
CT 06520-8114, USA; e-mail: osti@laplace.csb.yale.edu
† Max-Planck-Institut fur Biophysik, Abteilung fur Molekulare

Membranbiologie, Heinrich-Hoffmann-Strasse 7, 60528
Frankfurt/Main, Germany; e-mail: michel@mpibp-frankfurt.mpg.de
CRYSTALLIZATION OF INTEGRAL MEMBRANE PROTEINS SOLUBILIZED
                  IN DETERGENT MICELLES

  CRYSTALS STABILIZED MAINLY BY POLAR INTERACTIONS
       BETWEEN PROTEIN MOLECULES AND BETWEEN DETERGENT
       MOLECULES.

  DETERGENT MOLECULES MUST FIT INTO CRYSTAL LATTICE; THUS
       THEIR SIZE (SMALLER IS BETTER) AND CHEMISTRY ARE
       IMPORTANT.

  ADDITION OF SMALL AMPHIPHILES TO CRYSTALLIZATION MEDIUM
       OFTEN ENHANCES CRYSTAL FORMATION BY REPLACING
       THOSE DETERGENT MOLECULES THAT STERICALLY
       INTERFERE WITH LATTICE FORMATION. ALSO, BY MAKING
       MICELLES SMALLER, THEY CAN ALLOW BETTER CONTACT
       BETWEEN POLAR SURFACES OF PROTEIN.

  SMALL AMPHIPHILES ALSO INCREASE PROTEIN SOLUBILITY.
SEE: NOLLER ET AL., FEBS LETT. 504, 179-186 (2001) FOR DISCUSSION OF
MECHANISM OF CUBIC PHASE CRYSTALLIZATION
Proc. Natl. Acad. Sci. USA
Vol. 93, pp. 14532–14535, December 1996

Lipidic cubic phases: A novel concept for the crystallization of membrane proteins
EHUD M. LANDAU AND JŰRG P. ROSENBUSCH
Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
Communicated by H. Ronald Kaback, University of California, Los Angeles, CA, September 30,
1996 (received for review August 12, 1996)

ABSTRACT Understanding the mechanisms of action of membrane proteins requires the
elucidation of their structures to high resolution. The critical step in accomplishing
this by x-ray crystallography is the routine availability of well-ordered three-dimensional
crystals. We have devised a novel, rational approach to meet this goal using quasisolid
lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in
appropriate proportions, forms a structured, transparent, and complex three-
dimensional lipidic array, which is pervaded by an intercommunicating
aqueous channel system. Such matrices provide nucleation sites („„seeding‟‟)
and support growth by lateral diffusion of protein molecules in the membrane
(„„feeding‟‟). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases,
but not from micellar systems, implying a critical role of the continuity of the diffusion
space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted
to 3.7 Å resolution (NOW TO 1.6 D), with a space group P63, and unit cell dimensions
of a = b = 62 Å, c = 108 Å;  =  = 90º and  = 120º.
(HALORHODOPSIN ALSO CRYSTALLIZED IN THIS WAY.)
PNAS 96, 14706-14711 (1999)
Structural details of an interaction between cardiolipin and an integral membrane protein
Katherine E. McAuley* , Paul K. Fyfe‡ , Justin P. Ridge‡ , Neil W. Isaacs† , Richard J. Cogdell*,
and Michael R. Jones‡
*Division of Biochemistry and Molecular Biology and † Department of Chemistry, University of
Glasgow, Glasgow, G12 8QQ, United Kingdom; and ‡ Krebs Institute for Biomolecular Research
and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology,
University of Sheffield, Western Bank, Sheffield, S10 2UH, United Kingdom
Edited by Johann Deisenhofer, University of Texas Southwestern Medical Center, Dallas, TX, and
approved October 27, 1999 (received for review May 3, 1999)
           Anionic lipids play a variety of key roles in biomembrane function, including
providing the immediate environment for the integral membrane proteins that catalyze
photosynthetic and respiratory energy transduction. Little is known about the molecular
basis of these lipid–protein interactions. In this study, x-ray crystallography has been used
to examine the structural details of an interaction between cardiolipin and the photoreaction
center, a key light-driven electron transfer protein complex found in the cytoplasmic
membrane of photosynthetic bacteria. X-ray diffraction data col-lected over the resolution
range 30.0–2.1 Å show that binding of the lipid to the protein involves a combination of ionic
interactions between the protein and the lipid headgroup and van der Waals interactions
between the lipid tails and the electroneutral in-tramembrane surface of the protein. In the
headgroup region, ionic interactions involve polar groups of a number of
residues, the protein backbone, and bound water molecules. The lipid tails sit
along largely hydrophobic grooves in the irregular surface of the protein. In
addition to providing new information on the imme-diate lipid environment of a key integral
membrane protein, this study provides the first, to our knowledge, high-resolution x-ray
crystal structure for cardiolipin. The possible significance of this interaction between an
integral membrane protein and cardiolipin is considered.
PRINCIPLES OF MEMBRANE PROTEIN STRUCTURE [Scarlata,
"Membrane Protein Structure"; see also: White & Wimley, Ann. Rev.
Biophys. Biomol. Struct. 28, 319 (1999); White, in “Membranes”,
Biophysical Society on-line textbook].

 MEMBRANE PROTEIN ENVIRONMENT IS COMPLEX; IT INVOLVES
THE AQUEOUS REGION OUTSIDE MEMBRANE, ELECTRICAL
CHARGES AT THE MEMBRANE SURFACE, AND THE HYDROPHOBIC
INTERIOR OF THE MEMBRANE. THE STEEP DIELECTRIC GRADIENT
MAKES IT UNFAVORABLE TO BURY A CHARGE (20 kCAL/MOLE)
OR HAVE AN UNSATISFIED H-BOND (5 kCAL/MOLE); CONTROLS
WHICH RESIDUES INCORPORATE WITHIN THE MEMBRANE AND
WHICH REMAIN OUTSIDE, AS WELL AS SECONDARY AND
TERTIARY FOLDING (-HELICES AND -SHEETS FAVORED; LOOPS
AND RANDOM COILS DISFAVORED).

 LIPID HEAD GROUPS CAN HAVE STRONG ELECTROSTATIC AND H
BONDING INTERACTIONS WITH INTERFACIAL RESIDUES OF A
MEMBRANE PROTEIN.
 HYDROPHOBIC THICKNESS OF THE BILAYER MUST MATCH THE
HYDROPHOBIC LENGTH OF THE PROTEIN, e.g. TRANSMEMBRANE
HELIX MUST BE 18 RESIDUES LONG. BILAYER THICKNESS MAY
STABILIZE CERTAIN CONFORMATIONAL STATES.


 HYDROCARBON CHAIN PACKING MAY ALSO STABILIZE CERTAIN
PROTEIN STRUCTURES; FAVORS COMPONENTS WHICH DO NOT
GREATLY DISRUPT THEIR INTERACTIONS; e.g., PROTEIN
CYLINDRICAL SHAPES ARE PREFERRED.

 SOME GENERALIZATIONS: TERTIARY STRUCTURES OF
MEMBRANE PROTEINS HAVE SIMILAR PACKING AS SOLUBLE
PROTEINS; HELICES TILTED 20 TO ALLOW PACKING BETWEEN
SIDE CHAINS; H-BONDS BETWEEN HELICES ARE RARE AND SALT
BRIDGES NOT FOUND. BECAUSE OF HELIX DIPOLES,
ANTIPARALLEL ARRANGEMENT OF TRANSMEMBRANE HELICES
PREFERRED. TRP AND TYR MAINLY PRESENT AT INTERFACES; ACT
AS "ANCHORS".
       PROSTAGLANDIN H2 SYNTHASE-1

 INTEGRAL MEMBRANE PROTEIN, LOCATED PRIMARILY IN THE
ENDOPLASMIC RETICULUM.

 CATALYZES THE FIRST COMMITTED STEP IN PROSTAGLANDIN
BIOSYNTHESIS (ARACHIDONATE TO PROSTAGLANDIN H2).

 BIFUNCTIONAL: CYCLOOXYGENASE (TARGET FOR NSAID‟S:
ASPIRIN, IBUPROFEN, INDOMETHACIN); PEROXIDASE

 ANCHORED TO ONE LEAFLET OF BILAYER BY AMPHIPATHIC
HELICES.
                   PORINS

 FOUND IN OUTER MEMBRANES OF GRAM-NEGATIVE BACTERIA.

FORM WATER-FILLED CHANNELS THAT ALLOW THE
INFLUX/OUTFLUX OF SMALL HYDROPHILIC MOLECULES.

 HAVE TRIMERIC, BETA-BARREL STRUCTURES; RESIDUES
ALTERNATE BETWEEN FACING INWARD AND OUTWARD. THUS,
DO NOT HAVE LONG STRETCHES OF HYDROPHOBIC RESIDUES,
AS IN TRANSMEMBRANE HELICES.

PORES NARROWED BY INWARD FOLDING OF A LOOP INTO
LUMEN OF BARREL. HAVE WIDE ENTRANCE AND WIDE EXIT, AND
A SHORT CENTRAL CONSTRICTION (ABOUT 10 D DEEP AND 10 D
WIDE). MINIMIZES FRICTIONAL CONTACT WITH WALLS, WHILE
STILL EXCLUDING LARGE MOLECULES.
   A SIMPLIFIED OVERVIEW OF ELECTRON TRANSFER THEORY
ELECTRON TRANSFER (ET) IS A FUNDAMENTAL PROCESS IN
BIOLOGY, OCCURRING WITHIN AND BETWEEN PROTEIN MOLECULES
WHICH SERVE AS SCAFFOLDING FOR A VARIETY OF REDOX
CENTERS (METAL IONS, PORPHYRINS, FLAVINS, QUINONES, ETC.).

AMONG THE KEY QUESTIONS ARE:
1- HOW DO ELECTRONS MOVE OVER THE SOMETIMES LONG
DISTANCES BETWEEN REDOX CENTERS WHICH ARE IMPOSED BY
THE PROTEIN MATRIX (i.e. PATHWAYS)?
2- HOW DO DISTANCES BETWEEN REDOX CENTERS, FREE ENERGY
CHANGES FOR THE ET PROCESS, AND "SOLVENT" ENVIRONMENTS
OF THE REDOX CENTERS INFLUENCE ET RATES?
3- HOW DOES THE INTERVENING PROTEIN MATRIX INFLUENCE ET
RATES?
THE BACTERIAL PHOTOSYNTHETIC REACTION CENTER HAS
BECOME AN IMPORTANT MODEL SYSTEM FOR INVESTIGATING
THESE QUESTIONS (ITS STRUCTURE IS KNOWN, IT CONTAINS 8
REDOX CENTERS WHICH SPAN A DISTANCE OF APPROXIMATELY
80Å, AND ITS KINETIC PROPERTIES SPAN A TIME RANGE FROM
PICOSECONDS TO TENS OF SECONDS).
THE STARTING POINT FOR THEORETICAL TREATMENTS OF ET
REACTIONS IS THE FOLLOWING EQUATION (OBTAINED FROM TIME-
DEPENDENT QUANTUM MECHANICAL PERTURBATION THEORY):

kET = (4π2/h) VAB2 FC

WHERE VAB IS THE MATRIX ELEMENT FOR ELECTRONIC COUPLING
BETWEEN THE TWO REDOX SITES AND FC IS THE FRANCK-CONDON
(NUCLEAR) FACTOR.


VAB IS PROPORTIONAL TO THE OVERLAP OF THE ELECTRONIC
WAVEFUNCTIONS OF THE DONOR AND ACCEPTOR, AND IS THE
PRINCIPAL ORIGIN OF THE DISTANCE DEPENDENCE OF ET (ALSO
PROVIDES A ROLE FOR THE INTERVENING PROTEIN MATRIX).
SIMPLEST MODEL (NEGLECTING ROLE OF INTERVENING MEDIUM)
PREDICTS VAB PROPORTIONAL TO exp (-αR). APATHWAYS@ CONCEPT
PROPOSES THAT ELECTRONS TUNNEL BETWEEN LOCALIZED REDOX
CENTERS, UTILIZING BOTH THROUGH-BOND AND THROUGH-SPACE
ROUTES WHICH ARE HIGHLY SENSITIVE TO MOLECULAR STRUCTURE
(METHODS FOR CALCULATING THE EFFECTIVENESS OF THESE
ROUTES HAVE BEEN DEVELOPED).
FC ORIGINATES FROM THE REQUIREMENT (FRANCK-CONDON
PRINCIPLE) THAT THE NUCLEAR CONFIGURATION OF THE
REACTANTS MUST BE SUCH THAT THE ENERGY OF THE
REACTANTS AND PRODUCTS ARE EQUAL AT THE TRANSITION
STATE (THIS OCCURS VIA THERMAL FLUCTUATIONS AND/OR
VIBRATIONS; THIS PROVIDES A ROLE FOR PROTEIN DYNAMICS;
ENERGY TO ACHIEVE THIS CALLED "REORGANIZATION
ENERGY"), I.E. ET OCCURS BETWEEN STATES WHOSE NUCLEAR
COORDINATES DO NOT CHANGE. REORGANIZATION ENERGY IS
OFTEN DIVIDED BETWEEN CHANGES OCCURRING AT REDOX
CENTER (INNER SPHERE) AND THOSE OCCURRING IN
SURROUNDING PROTEIN/WATER MATRIX (OUTER SPHERE).

FC FACTOR CONTAINS THE DEPENDENCE OF ET RATE ON THE FREE
ENERGY CHANGE BETWEEN REACTANTS AND PRODUCTS AND ON
THE REORGANIZATION ENERGY.
THE SIMPLEST THEORETICAL TREATMENT OF FC FACTORS IS DUE
TO MARCUS (USING A CLASSICAL HARMONIC OSCILLATOR MODEL,
WHICH GENERATES PARABOLIC POTENTIAL ENERGY CURVES).
YIELDS THE WIDELY USED MARCUS EQUATION:

kET = (4π2/h) VAB2 [1/(4πλkT)1/2] exp [-(λ + ΔG0)2/4λkT]

WHERE λ = REORGANIZATION ENERGY.

ΔG0 = -RT ln Keq = -n F E0

WHERE F = FARADAY CONSTANT = 23.09 kcal/volt

ΔGI = (λ + ΔG0)2 / 4λ
 THE RELATIONSHIP BETWEEN kET AND ΔG0 IS SHOWN
 SCHEMATICALLY IN FOLLOWING GRAPHS. THIS YIELDS THE
 FOLLOWING PICTURE:
         AS THE DRIVING FORCE FOR ET INCREASES, THE RATE
 INCREASES AND THE ACTIVATION ENERGY DECREASES. WHEN λ =
 ΔG0, kET REACHES A MAXIMUM AND THE ACTIVATION ENERGY
 BECOMES ZERO. FURTHER INCREASES IN DRIVING FORCE
 RESULT IN A DECREASE IN REACTION RATE AND AN INCREASE IN
 ACTIVATION ENERGY (MARCUS INVERTED REGION).


ALTHOUGH MORE SOPHISTICATED TREATMENTS OF FC FACTORS
EXIST, OUR UNDERSTANDING OF THE WAYS IN WHICH
EXPERIMENTAL VARIABLES INFLUENCE REACTION RATES,
ESPECIALLY IN PROTEINS, IS OFTEN NOT SUFFICIENT TO JUSTIFY
USE OF MORE RIGOROUS THEORETICAL MODELS. THUS, FOR
EXAMPLE, TEMPERATURE CAN AFFECT ELECTRONIC COUPLING,
DRIVING FORCE, AND REORGANIZATIONAL ENERGY; SEVERAL
VIBRATIONAL MODES MAY BE COUPLED TO THE ET STEP; ETC.
   BACTERIAL PHOTOSYNTHETIC REACTION CENTER (R. viridis)

 CRYSTALLIZATION:
      AMMONIUM SULFATE PRECIPITATION IN PRESENCE OF LDAO
      AND HEPTANE-1,2,3-TRIOL.
 STRUCTURE:
      SUBUNITS (FOUR): L, M, H PLUS TIGHTLY-BOUND 4-HEME
      CYTOCHROME (c-TYPE; ABSENT IN SOME RC‟S).

 COFACTORS:
      BOUND BY L AND M SUBUNITS;
      4 BCHL, 2 BPHE, 2 QUINONES (QB SITE ONLY PARTLY
            OCCUPIED),1 CAROTENOID, 1 Fe (II);
      CAROTENOID AND QUINONE STRUCTURES VARY WITH
            SPECIES.

 LIPIDS:
       1 LDAO WELL-ORDERED IN CRYSTAL (NEAR QA BINDING
             SITE);
       CYTOCHROME SUBUNIT HAS A COVALENTLY LINKED
             DIGLYCERIDE (TO SULFUR OF C-TERMINAL
       CYSTEINE) WHICH EXTENDS INTO THE MEMBRANE.
 FUNCTION:
      MECHANISM OF ELECTRON TRANSFER

  ELECTRON FLOW ASYMMETRIC (ONLY VIA L PATHWAY).

  PHOTON ABSORPTION (OR ENERGY TRANSFER) RAISES P TO
 FIRST EXCITED SINGLET STATE (P*; NATURAL LIFETIME ~ 3 ns)
  ELECTRON TRANSFER TO Bph (HA) OCCURS IN ~ 3.5 ps; ROLE
 OF BRIDGING Bchl (BA) UNCERTAIN (i.e. DOES ELECTRON RESIDE
 HERE FOR ANY FINITE TIME, OR IS BA ONLY INVOLVED IN
 COUPLING BETWEEN P AND HA?). IT CLEARLY PLAYS A ROLE,
 SINCE RATE IS TOO FAST FOR DIRECT TRANSFER FROM P TO HA.
  ELECTRON TRANSFER TO QA OCCURS IN ~ 200 ps.
 ELECTRON TRANSFER TO QB OCCURS IN ~ 100 μs (Fe DOES NOT
PLAY A DIRECT ROLE IN TRANSFER; EXACT FUNCTION UNCLEAR;
MAY BE STRUCTURAL).
 QB PICKS UP TWO ELECTRONS AND TWO PROTONS AND
DISSOCIATES FROM BINDING SITE (~ 5 ms; THIS IS RATE-LIMITING
STEP); SITE REFILLED FROM QUINONE POOL.

 REDUCED QB IS REOXIDIZED BY CYTOCHROME bc1;
ELECTRONS THEN TRANSFERRED TO CYTOCHROME c2 AND
THEN TO P+ (EITHER VIA 4-HEME CYTOCHROME OR DIRECTLY).

 OVERALL QUANTUM EFFICIENCY IS CLOSE TO UNITY.

 ROLE OF PROTEIN: GENERALLY THOUGHT THAT PROTEIN
MOTIONS ARE COUPLED TO ELECTRON TRANSFER (e.g.
VIBRATIONAL MODES; RELAXATIONS THAT STABILIZE VARIOUS
INTERMEDIATE STATES).
       PROTON TRANSPORT IN BACTERIORHODOPSIN
SIMPLEST KNOWN EXAMPLE OF A TRANSMEMBRANE ION PUMP.
   DARK ADAPTED STATE
 ACTIVE SITE CAN BE THOUGHT OF AS CONSISTING OF A
HIGHLY POLARIZED WATER MOLECULE (W402) COORDINATED
BY THE PROTONATED RETINAL SCHIFF BASE, WHICH IS SALT-
LINKED TO TWO ANIONIC ASP RESIDUES, ASP-85 AND ASP-212.
SCHIFF BASE IS DEPROTONATED DURING THE PHOTOCYCLE.

 2 CHANNELS LEAD FROM ACTIVE SITE TO SURFACE:

EXTRACELLULAR (HYDROPHILIC, WIDE); CONTAINS H-BONDED
NETWORK OF FOUR RESIDUES (ARG-82, TYR-57, GLU-194, GLU-
204), AND AT LEAST SIX BOUND WATER MOLECULES.

CYTOPLASMIC (HYDROPHOBIC, NARROW); CONTAINS ONLY ONE
RESIDUE INVOLVED IN PROTON TRANSPORT (ASP-96; HAS AN
UNUSUALLY HIGH pK), AND FEWER BOUND WATERS. TO
REPROTONATE THE SCHIFF BASE DURING THE PHOTOCYCLE,
THIS REGION HAS TO UNDERGO CONFORMATIONAL CHANGES,
ALLOWING WATER TO ENTER.
  EVENTS FOLLOWING LIGHT ABSORPTION
   RETINAL PHOTOISOMERIZATION IS COUPLED TO PROTEIN
CONFORMATION CHANGES. THIS IS THE RESULT OF A STERIC AND
ELECTROSTATIC CONFLICT OF THE CHROMOPHORE WITH ITS BINDING
SITE. RELAXATION OF THIS CONFLICT DRIVES THE THERMAL
REACTIONS OF THE PHOTOCYCLE.
   PROTON IS TRANSFERRED TO ASP-85 WITHIN ABOUT 50 S.
  PROTON MAY BE DERIVED FROM SCHIFF BASE (SUGGESTED THAT
  SCHIFF BASE pK DECREASES AND ASP pK INCREASES DUE TO
  CHANGES IN ENVIRONMENT: SCHIFF BASE N MOVES TO
  HYDROPHOBIC REGION AND H-BONDS FORM TO CARBOXYL
  GROUP); MAY BE HELPED BY A SMALL MOVEMENT OF HELIX C
  WHICH BRINGS THEM CLOSER TOGETHER. ALSO POSSIBLE THAT
  PROTON DERIVES FROM THE BOUND WATER MOLECULE,
  GENERATING HYDROXYL WHICH REMOVES PROTON FROM
  RETINAL.
   PROTONATION STATES OF ASP-85, GLU-204 AND GLU-194
  LINKED. TRANSFER OF PROTON TO ASP-85 CAUSES MOVEMENT OF
  ARG-82 TOWARDS BOTTOM OF CHANNEL. THIS CAUSES pK OF
  GLU-204 TO DECREASE; GLU-204 TRANSFERS PROTON TO GLU-194,
  WHICH RELEASES PROTON AT EXTRACELLULAR SURFACE.
  REPROTONATION OF SCHIFF BASE FROM CYTOPLASM REQUIRES
 THAT pK OFASP-96 BE LOWERED AND PROTON PATHWAY CREATED
 (PROBABLY VIA BOUND WATER).
  PROTEIN CONFORMATION CHANGE IN M INTERMEDIATE IS
 CAUSED BY RETINAL STRAIGHTENING; 13-METHYL PUSHES ON TRP-
 182, MOVING HELIX F. CAUSES pK OF ASP-96 TO DECREASE, DUE TO
 INCREASE IN HYDRATION OF CYTOPLASMIC CHANNEL; RESULTS IN
 PROTON TRANSFER TO SCHIFF BASE.
             DARK RE-ISOMERIZATION OF RETINAL
  CAUSES REVERSAL OF PROTEIN CONFORMATIONAL CHANGE.
 RESTORES THE HIGH pK OF ASP-96, LEADING TO REPROTONATION
 FROM CYTOPLASM.
  THIS CAUSES PROTON TRANSFER FROM ASP- 85 TO GLU-204 (VIA
 ARG-82 AND BOUND WATER MOLECULE), THEREBY COMPLETING
 THE PHOTOCYCLE.
            SUMMARY OF OVERALL MECHANISM
PROTON TRANSPORT OCCURS VIA ALTERNATING ACCESS BETWEEN
SCHIFF BASE AND THE TWO MEMBRANE SURFACES. DIRECTION OF
TRANSFER IS CONTROLLED BY pK CHANGES CAUSED BY COUPLING
BETWEEN RETINAL PHOTOISOMERIZATION AND PROTEIN
CONFORMATIONAL CHANGES.

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:51
posted:8/3/2011
language:English
pages:37